Abstract
Normal phase, isocratic high-performance liquid chromatography methods are described for the separation of neutral lipid and fatty acid classes using low wavelength detection. Prior to high-performance liquid chromatography, methods were developed and are described for the separation of phospholipids from neutral lipids and fatty acids using small (600 mg) silica Sep-PaksTM. Recoveries of cholesteryl esters, triglycerides, fatty acids, and phospholipids from the silica columns were greater than 95%. Two mobile phases are described for lipid class separation by high-performance liquid chromatography. The first mobile phase, hexane-2-propanol-acetic acid 100:0.5:01, resulted in incomplete separation of cholesteryl ester and triglyceride but excellent separations of fatty acids and cholesterol. The second mobile phase, hexane-n-butyl chloride-acetonitrile-acetic acid 90:10:1.5:0.01, resulted in complete separation of the four lipid classes. This mobile phase also separated individual triglycerides and fatty acids based on the number of double bonds. Recoveries of radiolabeled lipids for the four lipid classes from high-performance liquid chromatography was greater than 95% with both mobile phases.
Highlights
All samples for high-performance liquid chromatography (HPLC) separations were dissolved in the solvent that was used as the mobile phase
Lipid extracts from the Bligh and Dyer extraction procedure were evaporated to dryness under nitrogen, taken up in 2.0 ml of chloroform-acetic acid 100:1 and applied to silica Sep-PakTMcolumns for separation of phospholipids from neutral lipids and fatty acids
Recoveries of lipid utilizing this isocratic mobile phase were excellent, being greater than 95% for all lipid classes (Table 2)
Summary
Lipids were extracted from rat, rabbit, and human sera, from rat liver, and from cultured rat aortic smooth muscle cells by the method of Bligh and Dyer [5]. After 24 hr, the rat was killed and the liver (16.2 g) was homogenized and extracted using the Bligh and Dyer method [5].The phospholipidfraction was obtained by separation from neutral lipid and fatty acids using a silica SepPakTMcolumn (see below). HPLC was performed using a Waters Associates (Milford, MA) Model 660 solvent programmer with two Model 6000 A solvent delivery pumps, a model U6K injector, and a model 450 variable wavelength detector operated at 206 nm. The mobile phase flow rate was 2.0 ml/min. All samples for HPLC separations were dissolved in the solvent that was used as the mobile phase. The HPLC system was maintained at room temperature, approximately 22°C
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